Total synthesis of odoamide, a novel cyclic depsipeptide, from an Okinawan marine cyanobacterium

Article abstract of DOI:10.1039/c6ob01583b

Odoamide is a novel cyclic depsipeptide with highly potent cytotoxic activity isolated from the Okinawan marine cyanobacterium Okeania sp. It contains a 26-membered macrocycle composed of a fatty acid moiety, a peptide segment and isoleucic acid. Four pos

Full text of DOI:10.1039/c6ob01583b

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Total synthesis of odoamide, a novel cyclic depsipeptide from an Okinawan marine
DOI: 10.1039/C6OB01583B
cyanobacterium
Masato Kanedaa, Kosuke Sueyoshib, Toshiaki Teruyab, Hiroaki Ohnoa, Nobutaka Fujiia, Shinya
Oishia,*
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
b Faculty of Education, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan
*Corresponding Author:
Shinya Oishi, Ph.D.
Graduate School of Pharmaceutical Sciences
Kyoto University
Sakyo-ku, Kyoto, 606-8501, Japan
Tel: +81-75-753-4551; Fax: +81-75-753-4570
E-mail (S.O.): soishi@pharm.kyoto-u.ac.jp
1

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DOI: 10.1039/C6OB01583B
Abstract
Odoamide is a novel cyclic depsipeptide with highly potent cytotoxic activity isolated from the
Okinawan marine cyanobacterium Okeania sp. It contains a 26-membered macrocycle composed of
a fatty acid moiety, a peptide segment and an isoleucic acid. Four possible stereoisomers of the
odoamide polyketide substructure were synthesised using a chiral pool approach. The first total
synthesis of odoamide was also successfully achieved. The structure of synthetic odoamide was
verified by comparing its NMR spectra with those of the natural product.
2

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DOI: 10.1039/C6OB01583B
Introduction
Many peptide secondary metabolites derived from natural resources show attractive biological
activities.1 Because of their favourable drug-like properties including good membrane permeability
and biostability,2 a number of synthetic and medicinal chemistry studies of macrocyclic peptides and
highly N-methylated peptides have been carried out.3 Among them, aurilide-class cyclic
depsipeptides possess highly potent antiproliferative activity against cancer cell lines (Figure 1). The
first 26-membered cyclic depsipeptide, aurilide (1a), was isolated from the sea hare Dolabella
auricularia.4 The related depsipeptides, aurilide B (1b) and C (1c), from Lyngbya majuscula also
show potent cytotoxicity.5 Kulokekahilide-2 (2) is a similar cytotoxic depsipeptide from a marine
mollusk, Philinopsis speciosa, which exhibits two conformers of the 26-membered macrocycle in
dichloromethane.6 Lagunamide A (3a) and B (3b) from Lyngbya majuscula has antimalarial activity
against Plasmodium falciparum at submicromolar concentrations.7 Lagunamide C (3c)8 and
palau’amide (4)9 exhibit comparable cytotoxicity at nanomolar concentrations to other aurilide-class
depsipeptides, although these peptides have unique 27-membered and 24-membered macrocycles,
respectively. The configurations of the component amino acids of the depsipeptides were investigated
by chiral HPLC, chiral GC-MS, and Marfey’s analyses,10 while the stereoselective synthesis and the
NMR analysis facilitated the determination of the absolute stereochemistries of the fatty acid
substructure. In some cases, the structure was verified or revised through synthetic studies of natural
products and their stereoisomers.11
Odoamide (5) is a novel cyclic depsipeptide from the Okinawan marine cyanobacterium Okeania
sp. (Figure 2A), which shows highly potent cytotoxic activity against HeLa S3 cell lines.12 The overall
structure of the 26-membered macrocycle is similar to those of aurilide-class depsipeptides, and
comprises three substructures: a fatty acid moiety, a peptide segment (Ala-D-MePhe-Sar-Ile-MeAla)
and an isoleucic acid. At the initial stage of this study, the absolute configurations of the constituent
amino acids and isoleucic acid in 5 were determined by chiral HPLC analysis and Marfey’s analysis.
The absolute configuration of the 5-hydroxy group of the polyketide part was determined by Mosher’s
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DOI: 10.1039/C6OB01583B
method, while the remaining configurations of the polyketide were ambiguous. In this study, we
carried out a synthetic study of odoamide to verify its structure and complete stereochemistry.
The synthetic strategy is illustrated in Scheme 1. During the cyclisation of the linear peptide,
epimerisation and dimer formation are often problematic.3e,3f,14 To avoid the less reactive process of
N-methylated amide (CO-NMe) or ester bond formation compared with standard peptide bond (CO-
NH) formation, we chose macrocyclisation of the Ala and D-allo-isoleucic acid residues of linear
precursor 6 for odoamide (5).11d Peptide 6 could be prepared by coupling of alcohol 7, MeAla 8 and
tetrapeptide 9, which could be obtained by standard solid-phase peptide synthesis. The alcohol 7
could be synthesised by coupling of D-allo-isoleucic acid ester 1115 with a carboxylic acid 10.
Results and Discussion
Synthesis of the polyketide substructure of odoamide
The stereochemistries of the polyketide part were unknown when we started this study. Therefore,
it was necessary to synthesise all the possible polyketide substructures in odoamide 5. The polyketide
substructure in lagunamide A (3a), a closely related structural analogue of 5, has 5S,7R-dihydroxy
and 6S,8S-dimethyl groups. Additionally, the aurilide-class depsipeptides 1a-c, 2, and 3a,b possess
the syn-1,3-diol moiety with a 5S-hydroxy configuration. On the basis of the structures of these related
molecules, we expected that the plausible stereochemical configuration of the natural odoamide 5
was 5S,6S,7R,8S. Among these four stereocentres, the configuration at the C8-methyl group was
ambiguous because attempts to determine it based on derivatisation and NMR analysis of odoamide
(5) were unsuccessful. It was also desirable to confirm the stereochemistry of the C6-methyl group.
Therefore, we designed four possible methyl esters 12a-d as polyketide substructure substrates
(Figure 2B).
The methyl esters 12a,b were synthesised from the (S)-Roche ester 13 according to a similar
process in previous reports by us and others (see supplementary information).11a,12 Preparation of
(5S,6R,7R,8S)-ester 12c and (5S,6R,7R,8R)-ester 12d started from the commercially available (R)-
4

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Roche ester ent-13 in a similar manner (Scheme 2). (R)-Roche ester ent-13 was converted to alcohol
DOI: 10.1039/C6OB01583B
ent-15 via benzyl protection16 and LiAlH4-mediated reduction. After Swern oxidation, a n-Bu2BOTf-
mediated Evans aldol reaction17 of the resulting aldehyde provided the syn-aldol products 16c and
17d. The requisite stereochemistries at the C8 chiral centre in 12c and 12d were generated at this step
by using propionyl- and pentanoyl-oxazolidinones, respectively. TBS protection of the secondary
alcohol in 16c and 17d followed by removal of the chiral auxiliary with LiBH4 gave alcohols 20c18
and 21d. Swern oxidation of 20c and the subsequent Wittig reaction of the resulting aldehyde with
ethylidene-triphenylphosphorane provided olefin 22c as an E/Z isomeric mixture. Hydrogenation of
22c in the presence of Pd/C afforded the key alcohol 23c with a threo/threo-configuration. Separately,
tosylation of 21d followed by LiAlH4-mediated reduction afforded benzyl ether 24d, which was
converted to the corresponding alcohol 23d (with a threo/erythro-configuration) by hydrogenation.
Swern oxidation of 23c followed by a Mukaiyama aldol reaction19 with 1-methoxy-2-methyl-1-
trimethylsiloxy-1,3-butadiene (25)20 produced methyl ester 12c with a (5S)-hydroxy group (dr
>99:1).21 The ester 12d was obtained from 23d by the identical protocol.
The stereochemistry of 5-hydroxy group in alcohol 12a was confirmed by the NMR analysis of
the corresponding acetonide (Scheme 3). TBS deprotection of 26a22 and 12a provided 1,3-diols 27a
and 28a, which were treated with 2,2-dimethoxypropane in the presence of PPTS to give acetonides
13
29a and 30a, respectively. It is known that C NMR chemical shifts of the ketal methyl groups in
syn- and anti-1,3-diol acetonides are different.23 A syn-acetonide shows different chemical shifts for
the two ketal methyl groups (e.g., 19.5 and 30.0 ppm for 30a) because of its predominant chair
conformation. In contrast, an anti-acetonide shows close chemical shifts (e.g., 23.5 and 25.2 ppm for
29a), because the anti-isomer exists in a twist-boat conformation to avoid the 1,3-diaxial interaction
that would be present in the chair conformation. Accordingly, it was demonstrated that 1,3-diol 28a,
the precursor of the acetonide 30a has the desired 1,3-syn configuration. Of note, esters 12a-d were
employed as the key substrates for the stereochemical assignment of the polyketide substructure in 5
in our previous research.12 Manipulations of esters 12a-d including DIBAL-mediated reductive
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transformation provided the corresponding triol derivatives. The comparative NMR analysis between
DOI: 10.1039/C6OB01583B
natural product-derived triol and synthetic triols demonstrated that the polyketide substructure had
the 5S,6S,7R,8S configuration (see supplementary information).12
Synthesis of odoamide and its biological evaluation
After the determination of the stereochemistry of the polyketide part,12 we attempted the total
synthesis of odoamide using the (5S,6S,7R,8S)-ester 12a. Synthesis of odoamide (5) began with
methylthiomethyl (MTM) protection of the secondary hydroxy group in 12a to give thioacetal 31
(Scheme 4).11a,24 Hydrolysis of 31 with LiOH followed by coupling with D-allo-isoleucic acid
phenacyl ester (11) with 2-methyl-6-nitrobenzoic anhydride (MNBA)25 and DMAP afforded ester 32.
The TBS group in 32 was deprotected with HF·pyridine to produce the corresponding alcohol 7. In
the coupling of Fmoc-MeAla 8 with 7 using DCC and DMAP, significant epimerisation occurred.
The coupling using Fmoc-MeAla-Cl26 with 7 in the presence of DIPEA followed by Fmoc
deprotection with Et2NH gave amine 33 in 54% yield (two steps) without epimerisation. The
tetrapeptide 9 was conjugated with 33 using EDCI-HOAt to afford 34 as a 1.4:1 epimeric mixture at
the -position of Ile.27 After removal of the phenacyl (with Zn and AcOH) and Fmoc groups (with
Et2NH), the epimer mixture of the linear peptides was separated into the desired 6a (major, L-Ile) and
undesired 6b (minor, D-allo-Ile) by HPLC purification. Cyclisation of 6a and 6b with HATU followed
by deprotection of the MTM group with AgNO3 and 2,6-lutidine gave the desired odoamide 5a and
its diastereomer 5b. Both cyclisations of 6a and 6b proceeded smoothly within five hours without
epimarisation. The configurations of L-Ile and D-allo-Ile in peptides 5a and 5b, respectively, were
determined by Marfey analysis and 1H NMR analysis after acid hydrolysis.
We analysed the 1H NMR and 13C NMR spectra of the natural and synthetic products (Figures 3
and 4). The NMR spectra of the synthetic odoamide 5a were identical with those of the natural product
5, suggesting the chemical structure of odoamide was the same as 5a. The cytotoxicity of synthetic
5a and 5b against A549 cells was also evaluated by the MTS assay. Peptide 5a showed highly potent
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cytotoxicity (IC50 = 2.1 nM), corroborating our correct structural assignment ofDoOdI: o10a.1m03i9d/Ce6O(B50)1.583B
However, the epimer peptide 5b showed significantly less potent antiproliferative activity (IC50
0.54 M), suggesting that the L-Ile configuration is crucial for the cytotoxic activity of odoamide.
=
Conclusions
In this study, the total synthesis of odoamide was completed via the synthesis of four possible
polyketide substructures 12a-d. The NMR spectra of the synthetic peptide 5a were identical with
those of the natural odoamide 5. Accordingly, the full structural assignment and first total synthesis
of odoamide were achieved.
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DOI: 10.1039/C6OB01583B
Experimental Section
Synthetic General Method
NMR spectra were recorded using a JEOL ECA-500 spectrometer. Chemical shifts are reported in δ
(ppm) relative to Me4Si (in CDCl3) as an internal standard. 13C NMR spectra were referenced to the
residual solvent signal. Melting points were measured by a hot stage melting points apparatus
(uncorrected). Exact mass (HRMS) spectra were recorded on a Shimadzu LC-ESI-IT-TOF-MS
equipment. IR spectra were determined on a JASCO FT/IR-4100 spectrometer. Optical rotations were
measured with a JASCO P-1020 polarimeter. For flash chromatography, Wakogel C-300E (Wako)
was employed. For analytical HPLC, a Cosmosil 5C18-ARII column (4.6  250 mm, Nacalai Tesque,
Inc.) was employed with a linear gradient of CH3CN (with 0.1% (v/v) TFA, except for the analysis
of final products 5a,b using solvents without TFA) in H2O at a flow rate of 1 cm3 min-1, and eluting
products were detected by UV at 220 nm. Preparative HPLC was performed using a Cosmosil 5C18-
ARII preparative column (20  250 mm, Nacalai Tesque, Inc.) at a flow rate of 8 cm3 min-1. The
purity of the peptides 5a,b were determined by HPLC analysis (>95%). The synthetic procedures for
esters 12a,b were described in our previous report.12
Methyl (R)-3-benzyloxy-2-methylpropanoate (ent-14). To a stirred solution of ent-13 (9.9 g, 83.8
mmol) in CH2Cl2 (210 cm3) under argon were added benzyl 2,2,2-trichloroacetimidate (17.1 cm3,
92.2 mmol) in cyclohexane (420 cm3) and triflic acid (3.0 cm3, 33.5 mmol) at 0 °C. After 10 min, the
reaction mixture was warmed to room temperature and stirred for 18 h. The precipitated
trichloroacetamide was filtered off. The filtrate was washed with aqueous saturated NaHCO3 and
brine, dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was
purified by flash chromatography over silica gel with hexane–EtOAc (50:1 to 10:1) to give compound
ent-14 (14.8 g, 85%) as a colorless oil. The spectral data were in good agreement with those
previously reported.28
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(S)-3-Benzyloxy-2-methylpropan-1-ol (ent-15). To a stirred suspension of LiAlHD4O(I:410.0.103g9,/C160O5B0.19583B
mmol) in THF (175 cm3) under argon was added dropwise a solution of ent-14 (14.7 g, 70.6 mmol)
in THF (175 cm3) at 0 °C. After stirring for 1 h, the reaction mixture was poured into a saturated
aqueous solution of sodium potassium tartrate at 0 °C and stirred overnight at room temperature. The
whole was extracted with Et2O and the extract was washed with brine, dried over MgSO4. The filtrate
was concentrated under reduced pressure and the residue was purified by flash chromatography over
silica gel with hexane–EtOAc (9:1 to 3:1) to give compound ent-15 (10.3 g, 81%) as a colorless oil.
The spectral data were in good agreement with those previously reported.28
(R)-4-Benzyl-3-[(2R,3S,4R)-5-benzyloxy-3-hydroxy-2,4-dimethylpentanoyl]oxazolidin-2-one
(16c). To a stirred solution of oxalyl chloride (0.32 cm3, 3.72 mmol) in CH2Cl2 (7.4 cm3) under argon
was added DMSO (0.53 cm3, 7.44 mmol) in CH2Cl2 (1.2 cm3) at –78 °C. After stirring for 30 min, a
solution of ent-15 (334.7 mg, 1.86 mmol) in CH2Cl2 (6.4 cm3) was added dropwise and stirred at –
78 °C for 1 h. i-Pr2NEt (1.62 cm3, 9.3 mmol) was added and the reaction mixture was stirred at 0 °C
for 30 min. The mixture was quenched with aqueous saturated NH4Cl. The whole was extracted with
CH2Cl2 and the extract was washed with brine, dried over MgSO4. The filtrate was concentrated under
reduced pressure to give the corresponding aldehyde, which was used without further purification.
To a stirred solution of (R)-4-benzyl-3-propionyloxazolidin-2-one (429.2 mg, 1.84 mmol) in CH2Cl2
(9.2 cm3) under argon were added n-Bu2BOTf (1.0 M in CH2Cl2; 2.0 cm3, 2.00 mmol) and i-Pr2NEt
(0.38 cm3, 2.17 mmol) at –78 °C. After stirring for 1 h, the reaction mixture was warmed to 0 °C and
stirred for 30 min. To this solution was added the above aldehyde in CH2Cl2 (3.9 cm3) at –78 °C.
After stirring for 1 h, the mixture was warmed to –10 °C and stirred for 1 h. The mixture was quenched
with pH 7.0 phosphate buffer solution (1.8 cm3) and 30% H2O2 in MeOH (1:2, 4.2 cm3) and stirred
overnight at room temperature. The whole was concentrated under reduced pressure and extracted
with CH2Cl2. The extract was washed with aqueous saturated NaHCO3, dried over MgSO4. The
filtrate was concentrated under reduced pressure and the residue was purified by flash
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chromatography over silica gel with hexane–EtOAc (5:1 to 3:1) to give compound D1O6Ic: 10(.6101309/.C76OmB0g1,583B
80%, dr >15:1) as a colorless oil. The minor isomer was removed by column chromatography: []29
D
–43.2 (c 0.72, CHCl3); IR (neat) max/cm-1: 3504 (OH), 1779 (C=O); 1H NMR (500 MHz, CDCl3) δ:
1.05 (3H, d, J 6.9), 1.33 (3H, d, J 6.3), 1.87-1.93 (1H, m), 2.77 (1H, dd, J1 13.2, J2 9.7), 3.00 (1H, d,
J 2.9), 3.25 (1H, dd, J1 13.2, J2 3.2), 3.46-3.52 (2H, m), 3.96-4.03 (2H, m), 4.16-4.21 (2H, m), 4.51
13
(2H, s), 4.65-4.69 (1H, m), 7.20-7.21 (2H, m), 7.26-7.36 (8H, m); C NMR (125 MHz, CDCl3) δ:
12.4, 12.8, 36.2, 37.7, 40.5, 55.1, 66.0, 73.3, 73.9, 74.1, 127.4 (2C), 127.5 (2C), 128.3 (2C), 128.9
(2C), 129.4 (2C), 135.0, 138.1, 152.7, 177.0; HRMS (ESI) calcd for C24H29NNaO5 (MNa+):
434.1938; found: 434.1938.
(R)-4-Benzyl-3-[(2R,3S,4R)-5-benzyloxy-3-(tert-butyldimethylsilyloxy)-2,4-dimethylpentano-
yl]oxazolidin-2-one (18c). To a stirred solution of 16c (14.1 g, 34.3 mmol) in CH2Cl2 (137 cm3)
under argon were added TBSOTf (9.5 cm3, 41.2 mmol) and 2,6-lutidine (7.9 cm3, 68.6 mmol) at 0 °C.
The reaction mixture was warmed to room temperature and stirred for 2.5 h. The reaction was
quenched with 1N HCl. The whole was extracted with CH2Cl2 and the extract was washed with brine,
dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was purified
by flash chromatography over silica gel with hexane–EtOAc (9:1) to give compound 18c (16.0 g,
89%) as a colorless oil: []27D –38.2 (c 1.21, CHCl3); IR (neat) max/cm-1: 1780 (C=O); 1H NMR (500
MHz, CDCl3) δ: 0.04 (3H, s), 0.06 (3H, s), 0.90 (9H, s), 0.92 (3H, d, J 7.4), 1.25 (3H, d, J 6.9), 1.88-
1.93 (1H, m), 2.75 (1H, dd, J1 13.2, J2 9.7), 3.24 (1H, dd, J1 13.2, J2 3.2), 3.28 (1H, dd, J1 8.9, J2 7.2),
3.49 (1H, dd, J1 8.9, J2 6.6), 3.96-4.02 (1H, m), 4.08-4.16 (3H, m), 4.46-4.52 (2H, m), 4.59-4.64 (1H,
m), 7.20-7.34 (10H, m); 13C NMR (125 MHz, CDCl3) δ: –4.1, –3.9, 11.9, 15.0, 18.4, 26.1 (3C), 37.6,
38.8, 41.9, 55.4, 65.9, 72.8, 73.0, 73.4, 127.3, 127.4, 127.6 (2C), 128.3 (2C), 128.9 (2C), 129.4 (2C),
135.3, 138.6, 152.8, 175.9; HRMS (ESI) calcd for C30H43NNaO5Si (MNa+): 548.2803; found:
548.2808.
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DOI: 10.1039/C6OB01583B
(2S,3R,4R)-5-Benzyloxy-3-(tert-butyldimethylsilyloxy)-2,4-dimethylpentan-1-ol (20c). To a
stirred solution of 18c (22.4 g, 42.5 mmol) in THF (213 cm3) and MeOH (5.2 cm3, 127.6 mmol) under
argon was added LiBH4 (2.78 g, 127.6 mmol) at 0 °C. After stirring for 10 min, the reaction mixture
was warmed to room temperature. After 4 h, the mixture was cooled to 0 °C and quenched with
aqueous saturated NH4Cl. The whole was extracted with EtOAc and the extract was washed with
brine, dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue was
purified by flash chromatography over silica gel with hexane–EtOAc (9:1) to give compound 20c
1
(10.3 g, 69%) as a colorless oil: []29 –0.41 (c 1.23, CHCl3); IR (neat) max/cm-1: 3422 (OH); H
D
NMR (500 MHz, CDCl3) δ: 0.03 (3H, s), 0.08 (3H, s), 0.85 (3H, d, J 7.4), 0.89 (9H, s), 0.96 (3H, d,
J 6.9), 1.93-1.98 (1H, m), 1.99-2.05 (1H, m), 2.32 (1H, br s), 3.26 (1H, dd, J1 9.2, J2 6.3), 3.39 (1H,
dd, J1 9.2, J2 7.2), 3.47-3.51 (1H, m), 3.64-3.68 (1H, m), 3.88-3.89 (1H, m), 4.46-4.51 (2H, m), 7.26-
7.36 (5H, m); 13C NMR (125 MHz, CDCl3) δ: –4.5, –4.2, 12.8, 12.9, 18.2, 26.0 (3C), 35.9, 40.1, 66.3,
72.9, 73.6, 74.4, 127.5 (3C), 128.3 (2C), 138.5; HRMS (ESI) calcd for C20H36NaO3Si (MNa+):
375.2326; found: 375.2324.
(4S,5R,6R)-7-Benzyloxy-5-(tert-butyldimethylsilyloxy)-4,6-dimethylhept-2-ene (22c). To
a
stirred solution of oxalyl chloride (0.11 cm3, 1.30 mmol) in CH2Cl2 (6.5 cm3) under argon was added
DMSO (0.18 cm3, 2.60 mmol) in CH2Cl2 (0.43 cm3) at –78 °C. After stirring for 30 min, a solution
of 20c (228.9 mg, 0.65 mmol) in CH2Cl2 (2.2 cm3) was added dropwise and stirred at –78 °C for 1.5
h. i-Pr2NEt (0.57 cm3, 3.25 mmol) was added and the reaction mixture was stirred at 0 °C for 30 min.
The mixture was quenched with aqueous saturated NH4Cl. The whole was extracted with CH2Cl2 and
the extract was washed with brine, and dried over MgSO4. The filtrate was concentrated under
reduced pressure to give the corresponding aldehyde, which was used without further purification. To
a stirred suspension of ethyltriphenylphosphonium bromide (508.6 mg, 1.37 mmol) in THF (5.5 cm3)
under argon was added n-BuLi (1.6 mol dm-3 in hexane; 0.81 cm3, 1.30 mmol) at room temperature.
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After stirring for 30 min, a solution of the above aldehyde in THF (1.3 cm ) wasDaOdI: d10e.1d039a/nCd6OBth01e583B
reaction mixture was stirred for 1.5 h. The mixture was quenched with aqueous saturated NaHCO3.
The whole was extracted with EtOAc and the extract was washed with brine, and dried over MgSO4.
The filtrate was concentrated under reduced pressure and the residue was filtrated through a short pad
of silica gel with hexane–EtOAc (9:1). Further purification by flash chromatography over silica gel
with hexane–CHCl3 (8:1) gave compound 22c as a diastereomixture (163.7 mg, 69%, Z/E =7:1):
colorless oil; []27D +12.5 (c 1.17, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 0.01 (0.4H, s), 0.02 (2.6H,
s), 0.04 (0.4H, s), 0.05 (2.6H, s), 0.84-0.86 (3H, m), 0.89 (1.2H, s), 0.90 (7.8H, s), 0.94-0.97 (3H, m),
1.61 (2.6H, dd, J1 6.9, J2 1.7), 1.63 (0.4H, d, J 4.6), 1.96-2.03 (1H, m), 2.23-2.30 (0.1H, m), 2.60-
2.65 (0.9H, m), 3.22 (1H, dd, J1 8.9, J2 6.9), 3.40 (1H, dd, J1 8.9, J2 7.7), 3.56 (1H, dd, J1 8.0, J2 1.7),
4.43-4.52 (2H, m), 5.16-5.21 (1H, m), 5.35-5.42 (1H, m), 7.25-7.34 (5H, m); 13C NMR (125 MHz,
CDCl3) δ: –4.1, –4.0, –3.6, –3.5, 10.8, 11.1, 13.0, 17.6, 18.1, 18.3, 18.4, 18.5, 26.2 (6C), 35.9, 36.6,
37.1, 41.5, 72.7, 72.8, 73.6, 73.9, 76.0, 76.2, 122.7, 123.8, 127.4, 127.5 (2C), 128.3 (2C), 134.5, 134.9,
138.7; HRMS (ESI) calcd for C22H38NaO2Si (MNa+): 385.2533; found: 385.2534.
(2R,3R,4S)-3-(tert-Butyldimethylsilyloxy)-2,4-dimethylheptan-1-ol (23c). To a stirred solution of
22c (1.3 g, 3.7 mmol) in EtOH (37.0 cm3) was added 10% Pd/C (787.5 mg, 0.7 mmol) at room
temperature and the mixture was flushed with H2 gas (1 atm). After stirring for 1 h, the reaction
mixture was filtered through Celite. The filtrate was concentrated under reduced pressure and the
residue was purified by flash chromatography over silica gel with hexane–EtOAc (10:1) to give
compound 23c (938.1 mg, 92%) as a colorless oil: []28D –12.7 (c 1.13, CHCl3); IR (neat) max/cm-1:
3328 (OH); 1H NMR (500 MHz, CDCl3) δ: 0.06 (3H, s), 0.08 (3H, s), 0.85 (3H, d, J 6.9), 0.88-0.92
(15H, m), 1.09-1.16 (1H, m), 1.19-1.28 (1H, m), 1.31-1.41 (2H, m), 1.60-1.66 (1H, m), 1.90-1.97
13
(1H, m), 2.07 (1H, dd, J1 6.0, J2 4.3), 3.45-3.50 (1H, m), 3.62-3.67 (2H, m); C NMR (125 MHz,
CDCl3) δ: –4.2, –4.1, 12.6, 14.3, 15.8, 18.3, 20.8, 26.0 (3C), 35.9, 37.0, 39.5, 66.5, 77.4; HRMS (ESI)
calcd for C15H34NaO2Si (MNa+): 297.2220; found: 297.2221.
12

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DOI: 10.1039/C6OB01583B
Methyl (5S,6R,7R,8S,E)-7-(tert-butyldimethylsilyloxy)-5-hydroxy-2,6,8-trimethylundecen-2-
oate (12c). To a stirred solution of i-Pr2NH (0.16 cm3, 1.16 mmol) in THF (2.4 cm3) under argon was
added n-BuLi (2.6 mol dm-3 in hexane; 0.45 cm3, 1.16 mmol) at 0 °C. After 20 min, methyl tiglate
(0.13 cm3, 1.05 mmol) and TMSCl (0.20 cm3, 1.58 mmol) in THF (0.36 cm3) were added successively
at –78 °C. The stirring was continued for 1 h at this temperature and for additional 1.5 h at room
temperature. Then, pentane and cold saturated NaHCO3 were added to the reaction mixture. The
whole was extracted with pentane and the extract was washed with brine, and dried over MgSO4. The
filtrate was concentrated under reduced pressure to give compound 25, which was used without
further purification.20 To a stirred solution of oxalyl chloride (0.060 cm3, 0.70 mmol) in CH2Cl2 (3.5
cm3) under argon was added DMSO (0.099 cm3, 1.40 mmol) in CH2Cl2 (0.23 cm3) at –78 °C. After
stirring for 30 min, a solution of 23c (96.4 mg, 0.35 mmol) in CH2Cl2 (1.2 cm3) was added dropwise
and stirred at –78 °C for 1.5 h. i-Pr2NEt (0.49 cm3, 2.8 mmol) was added and the reaction mixture
was stirred at 0 °C for 30 min. The mixture was quenched with aqueous saturated NH4Cl. The whole
was extracted with CH2Cl2 and the extract was washed with brine, and dried over MgSO4. The filtrate
was concentrated under reduced pressure to give the corresponding aldehyde, which was used without
further purification. To a stirred solution of the above aldehyde in CH2Cl2 (2.7 cm3) and Et2O (0.27
cm3) under argon were added diene 25 and BF3·OEt2 (0.065 cm3, 0.53 mmol) at –78 °C. After stirring
for 2 h, a mixture of THF/H2O/1N HCl (5:1:0.4 v/v, 1.8 cm3) was added to the reaction mixture. The
mixture was warmed to room temperature and stirred for 15 min. Then, aqueous saturated NaHCO3
was added to the mixture at 0 °C. The whole was extracted with CH2Cl2 and the extract was washed
with brine, dried over MgSO4. The filtrate was concentrated under reduced pressure and the residue
was purified by flash chromatography over silica gel with hexane–EtOAc (20:1 to 10:1) to give
compound 12c (73.4 mg, 54%) as a colorless oil: []26D –22.4 (c 1.01, CHCl3); IR (neat) max/cm-1:
1
3523 (OH), 1716 (C=O); H NMR (500 MHz, CDCl3) δ: 0.08 (3H, s), 0.09 (3H, s), 0.85 (3H, d, J
6.9), 0.88-0.91 (12H, m), 0.94 (3H, d, J 6.9), 1.04-1.11 (1H, m), 1.15-1.22 (1H, m), 1.35-1.42 (1H,
13

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m), 1.46-1.53 (1H, m), 1.63-1.69 (2H, m), 1.87 (3H, s), 1.96 (1H, d, J 4.0), 2.31-2.42 (2H, m), 3.65-
DOI: 10.1039/C6OB01583B
3.66 (1H, m), 3.74 (3H, s), 3.79-3.83 (1H, m), 6.79-6.82 (1H, m); 13C NMR (125 MHz, CDCl3) δ: –
4.2, –3.5, 8.9, 12.7, 14.4, 15.4, 18.3, 21.1, 26.0 (3C), 34.7, 35.3, 37.9, 40.1, 51.7, 73.7, 78.8, 129.3,
138.8, 168.4; HRMS (ESI) calcd for C21H42NaO4Si (MNa+): 409.2745; found: 409.2748.
(2R,3R,4R)-1-Benzyloxy-3-(tert-butyldimethylsilyloxy)-2,4-dimethylheptane (24d). To a stirred
solution of 21d (7.3 g, 19.1 mmol) in CH2Cl2 (191 cm3) under argon were added Et3N (5.3 cm3, 38.2
mmol), TsCl (5.5 g, 28.7 mmol) and Me3N·HCl (1.8 g, 19.1 mmol) at room temperature. After stirring
for 1 h, the reaction was quenched with aqueous saturated NH4Cl. The whole was extracted with
CH2Cl2 and the extract was washed with brine, and dried over MgSO4. The filtrate was concentrated
under reduced pressure and the precipitated white solid was filtered off. The filtrate was concentrated
under reduced pressure to give the corresponding tosylate, which was used without further
purification. To a stirred suspension of LiAlH4 (2.2 g, 57.3 mmol) in THF (100 cm3) under argon was
added dropwise a solution of the above tosylate in THF (91 cm3) at 0 °C. After stirring for 10 min,
the reaction mixture was warmed to room temperature. After 5 h, the reaction mixture was poured
into a saturated solution of sodium potassium tartrate at 0 °C and stirred at room temperature for 1 h.
The whole was extracted with Et2O and the extract was washed with brine, dried over MgSO4. The
filtrate was concentrated under reduced pressure and the resulting residue was purified by flash
chromatography over silica gel with hexane–EtOAc (100:0 to 70:1) to give compound 24d (4.9 g,
70%) as a colorless oil: []27D +1.74 (c 1.16, CHCl3); 1H NMR (500 MHz, CDCl3) δ: 0.00 (3H, s),
0.03 (3H, s), 0.86-0.89 (18H, m), 1.00-1.05 (1H, m), 1.18-1.26 (1H, m), 1.36-1.42 (2H, m), 1.56-1.60
(1H, m), 1.95-2.00 (1H, m), 3.22 (1H, dd, J1 8.6, J2 6.6), 3.37 (1H, dd, J1 8.6, J2 7.4), 3.62 (1H, dd,
J1 5.7, J1 2.3), 4.45-4.52 (2H, m), 7.27-7.34 (5H, m); 13C NMR (125 MHz, CDCl3) δ: –4.3, –3.9, 11.9,
14.4, 15.9, 18.4, 20.7, 26.1 (3C), 35.5, 35.6, 38.3, 72.8, 74.4, 75.4, 127.4, 127.5 (2C), 128.3 (2C),
138.7; HRMS (ESI) calcd for C22H40NaO2Si (MNa+): 387.2690; found: 387.2691.
14

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Methyl (5S,6S,7R,8S,E)-7-(tert-butyldimethylsilyloxy)-2,6,8-trimethyl-5-(methylthiomethoxy)-
DOI: 10.1039/C6OB01583B
undec-2-enoate (31). To a stirred solution of 12a (2.3 g, 6.0 mmol) in DMSO (42.9 cm3) under argon
were added Ac2O (30.5 cm3) and AcOH (5.5 cm3) at room temperature. After stirring overnight, the
reaction mixture was cooled to 0 °C and quenched with aqueous saturated NaHCO3. The whole was
extracted with Et2O and the extract was washed with H2O and brine, and dried over MgSO4. The
filtrate was concentrated under reduced pressure and the residue was purified by flash
chromatography over silica gel with hexane–EtOAc (30:1) to give compound 31 (1.6 g, 59%) as a
1
colorless oil: []27 –75.4 (c 0.72, CHCl3); IR (neat) max/cm-1: 1716 (C=O); H NMR (500 MHz,
D
CDCl3) δ: 0.05 (6H, s), 0.85-0.92 (18H, m), 1.15-1.28 (2H, m), 1.33-1.42 (2H, m), 1.62-1.66 (1H, m),
1.85 (3H, d, J 1.1), 1.98-2.05 (1H, m), 2.16 (3H, s), 2.26-2.39 (2H, m), 3.47 (1H, dd, J1 6.9, J2 2.9),
13
3.73 (3H, s), 4.00-4.03 (1H, m), 4.53 (1H, d, J 11.5), 4.63 (1H, d, J 11.5), 6.92-6.95 (1H, m); C
NMR (125 MHz, CDCl3) δ: –3.7, –3.6, 11.4, 12.8, 14.0, 14.1, 14.3, 18.4, 20.9, 26.2 (3C), 29.1, 36.4,
36.6, 39.0, 51.6, 73.0, 75.9, 77.2, 128.4, 140.3, 168.5; HRMS (ESI) calcd for C23H46NaO4SSi (MNa+):
469.2778; found: 469.2779.
(2R,3S)-3-Methyl-1-oxo-1-(2-oxo-2-phenylethoxy)pentan-2-yl
(5S,6S,7R,8S,E)-7-(tert-butyl-
dimethylsilyloxy)-2,6,8-trimethyl-5-(methylthiomethoxy)undec-2-enoate (32). To a stirred
solution of 31 (1.14 g, 2.6 mmol) in MeOH (17 cm3) and THF (17 cm3) was added 1N LiOH (17 cm3)
at 0 °C. The reaction mixture was warmed to 30 °C and stirred overnight. The mixture was
concentrated under reduced pressure and EtOAc and 1N HCl were added to the residue. The whole
was extracted with EtOAc and the extract was washed with brine, and dried over MgSO4. The filtrate
was concentrated under reduced pressure and the residue was filtrated through a short pad of silica
gel with hexane-EtOAc (3:1) to give 10, which was used without further purification. To a stirred
solution of the acid 10 in CH2Cl2 (12.8 cm3) were added MNBA (1.32 g, 3.8 mmol), DMAP (935.0
mg, 7.7 mmol) and 11 (959.0 mg, 3.8 mmol) at room temperature. After stirring overnight, the mixture
was quenched with 1N HCl. The whole was extracted with CH2Cl2 and the extract was washed with
15

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brine, and dried over MgSO4. The filtrate was concentrated under reduced pressure DaOnId: 10t.h10e39r/eCs6iOdBu01e583B
was purified by flash chromatography over silica gel with hexane–EtOAc (10:1) to give compound
32 (1.48 g, 87%) as a colorless oil: []26D –43.5 (c 0.89, CHCl3); IR (neat) max/cm-1: 1710 (C=O);
1H NMR (500 MHz, CDCl3) δ: 0.05 (3H, s), 0.07 (3H, s), 0.86-0.91 (18H, m), 0.98 (3H, t, J 7.4),
1.15 (3H, d, J 6.9), 1.18-1.28 (2H, m), 1.34-1.47 (3H, m), 1.52-1.58 (1H, m), 1.62-1.64 (1H, m), 1.89
(3H, s), 1.99-2.03 (1H, m), 2.11 (3H, s), 2.19-2.25 (1H, m), 2.34-2.37 (2H, m), 3.47 (1H, dd, J1 7.4,
J2 2.9), 4.01-4.04 (1H, m), 4.52 (1H, d, J 11.5), 4.61 (1H, d, J 11.5), 5.19 (1H, d, J 3.4), 5.25 (1H, d,
J 16.6), 5.55 (1H, d, J 16.6), 7.04-7.07 (1H, m), 7.49 (2H, t, J 7.7), 7.60-7.63 (1H, m), 7.89-7.91 (2H,
13
m); C NMR (125 MHz, CDCl3) δ: –3.6, –3.5, 11.3, 11.8, 12.7, 14.1, 14.2 (2C), 14.3, 18.4, 20.9,
26.2 (3C), 26.3, 29.4, 36.4, 36.6, 36.9, 39.2, 66.2, 73.0, 74.6, 76.1, 77.2, 127.7 (2C), 128.2, 128.9
(2C), 133.9, 134.1, 141.4, 167.4, 169.7, 191.6; HRMS (ESI) calcd for C36H60NaO7SSi (MNa+):
687.3721; found: 687.3720.
(2R,3S)-3-Methyl-1-oxo-1-(2-oxo-2-phenylethoxy)pentan-2-yl
(5S,6R,7R,8S,E)-7-hydroxy-
2,6,8-trimethyl-5-(methylthiomethoxy)undec-2-enoate (7). To a stirred solution of 32 (80.7 mg,
0.12 mmol) in THF (0.80 cm3) and pyridine (0.20 cm3) was added HF·pyridine (0.50 cm3) at 0 °C.
The reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture
was poured into aqueous saturated NaHCO3 at 0 °C. The whole was extracted with EtOAc, and the
extract was washed with brine, 1N HCl and brine, and dried over MgSO4. The filtrate was
concentrated under reduced pressure and the residue was purified by flash chromatography over silica
gel with hexane–EtOAc (6:1) to give compound 7 (49.2 mg, 74%) as a colorless oil: []25D –5.29 (c
1.08, CHCl3); IR (neat) max/cm-1: 3526 (OH), 1708 (C=O); 1H NMR (500 MHz, CDCl3) δ: 0.83-0.85
(6H, m), 0.89-0.91 (3H, m), 0.98 (3H, t, J 7.4), 1.15 (3H, d, J 6.9), 1.24-1.37 (4H, m), 1.38-1.46 (1H,
m), 1.50-1.59 (1H, m), 1.61-1.64 (1H, m), 1.90 (3H, s), 1.92-1.99 (1H, m), 2.15-2.16 (4H, m), 2.19-
2.26 (1H, m), 2.37-2.49 (2H, m), 3.38-3.41 (1H, m), 4.08-4.12 (1H, m), 4.64 (2H, s), 5.22 (1H, d, J
16

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2.9), 5.26 (1H, d, J 16.6), 5.55 (1H, d, J 16.6), 7.03-7.06 (1H, m), 7.49 (2H, t, J 7.7),D7O.I6: 100.-1703.96/3C6(O1BH01,583B
m), 7.89-7.91 (2H, m); 13C NMR (125 MHz, CDCl3) δ: 11.4, 11.7 (2C), 12.6, 14.2 (3C), 20.5, 26.3,
29.6, 34.4, 36.7, 36.9, 38.5, 66.2, 73.5, 74.6, 76.2, 78.3, 127.7 (2C), 128.4, 128.9 (2C), 133.9, 134.1,
140.7, 167.4, 169.7, 191.6; HRMS (ESI) calcd for C30H46NaO7S (MNa+): 573.2856; found: 573.2855.
(2R,3S)-3-Methyl-1-oxo-1-(2-oxo-2-phenylethoxy)pentan-2-yl (5S,6S,7R,8S,E)-2,6,8-trimethyl-
7-[(N-methyl-L-alanyl)oxy]-5-(methylthiomethoxy)undec-2-enoate (33). Fmoc-MeAla-Cl was
synthesised by the identical procedure reported previously.26 To a stirred solution of Fmoc-MeAla-
OH (227.7 mg, 0.70 mmol) in CH2Cl2 (3.9 cm3) were added DMF (0.0054 cm3, 0.070 mmol) and
SOCl2 (0.508 cm3, 7.0 mmol) at room temperature. After stirring for 1 h, the mixture was concentrated
under reduced pressure to give Fmoc-MeAla-Cl, which was used without further purification. To a
stirred solution of 7 (152.6 mg, 0.28 mmol) and the above Fmoc-MeAla-Cl in 1,2-dichloroethane (2.8
cm3) was added (i-Pr)2NEt (0.244 cm3, 1.40 mmol) at room temperature. The reaction mixture was
warmed to 40 °C and stirred for 14 h. The mixture was cooled to room temperature and quenched
with saturated aqueous NH4Cl. The whole was extracted with CH2Cl2 and the extract was washed
with washed with brine, and dried over MgSO4. The filtrate was concentrated under reduced pressure
and the residue was filtrated through a short pad of silica gel with hexane–EtOAc (9:1 to 3:1) to give
crude Fmoc-protected amine, which was used without further purification. To a stirred solution of the
above protected amine in MeCN (7.0 cm3) was added Et2NH (2.3 cm3) at 0 °C. The reaction mixture
was warmed to room temperature and stirred for 1.5 h. The mixture was concentrated under reduced
pressure and the residue was purified by flash chromatography over silica gel with hexane–EtOAc
(3:1 to 1:2) to give compound 33 (94.9 mg, 54%) as a yellow oil: []27D –55.4 (c 0.79, CHCl3); IR
(neat) max/cm-1: 1712 (C=O); 1H NMR (500 MHz, CD3CN) δ: 0.78 (3H, t, J 7.2), 0.81-0.84 (6H, m),
0.86 (3H, t, J 7.4), 0.99 (3H, d, J 6.9), 1.02-1.08 (1H, m), 1.09-1.14 (1H, m), 1.16 (3H, d, J 7.1), 1.23-
1.34 (3H, m), 1.39-1.47 (1H, m), 1.69-1.77 (4H, m), 1.95 (3H, s), 2.02-2.08 (1H, m), 2.13-2.17 (1H,
m), 2.19-2.27 (4H, m), 2.32-2.36 (1H, m), 3.13 (1H, q, J 7.1), 3.63 (1H, dt, J1 10.3, J2 2.6), 4.44 (1H,
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d, J 11.5), 4.55 (1H, d, J 11.5), 4.79 (1H, dd, J1 10.3, J2 2.3), 5.05 (1H, d, J 3.4), 5.30D(O1IH: 10,.1d0,39J/C166O.B60)1,583B
5.42 (1H, d, J 16.6), 6.78-6.81 (1H, m), 7.46 (2H, t, J 8.0), 7.57-7.61 (1H, m), 7.85-7.87 (2H, m); 13C
NMR (125 MHz, CD3CN) δ 10.3, 12.0, 12.8, 13.1, 14.1, 14.4, 14.6, 18.7, 20.9, 26.8, 29.6, 34.4, 34.5,
36.7, 37.0, 37.7, 59.1, 67.7, 73.5, 75.3, 76.1, 78.3, 128.7 (2C), 129.0, 129.8 (2C), 134.9, 135.0, 142.4,
167.9, 170.5, 174.9, 193.2; HRMS(FAB) calcd for C34H54NO8S (MH+): 636.3565; found: 636.3569.
Linear peptides (6a,b). To a stirred solution of 33 (59.5 mg, 0.094 mmol), peptide 9 (185.2 mg) and
HOAt (38.4 mg, 0.28 mmol) in CH2Cl2 (3.1 cm3) was added EDCI·HCl (54.1 mg, 0.28 mmol) at 0 °C.
The reaction mixture was warmed to room temperature and stirred for 24 h. The mixture was
quenched with aqueous saturated NaHCO3. The whole was extracted with CH2Cl2 and the extract was
washed with aqueous saturated NH4Cl, dried over MgSO4. The filtrate was concentrated under
reduced pressure and the residue was purified by flash chromatography over silica gel with hexane–
EtOAc (1:1 to 2:3) to give peptide 34 as a 1.4:1 diastereomixture, which was used without further
purification. To a stirred solution of 34 in AcOH/EtOAc/H2O (60:35:5, 4.3 cm3) was added Zn (92.2
mg, 1.4 mmol) at room temperature. After stirring for 8 h, the reaction mixture was filtered through
Celite, and 1N HCl was added to the filtrate. The whole was extracted with EtOAc and the extract
was washed with brine, dried over MgSO4. After the filtrate was concentrated under reduced pressure,
AcOH was removed by azeotropic distillation with toluene to give the corresponding carboxylic acid,
which was used without further purification. To a stirred solution of the above acid in MeCN (2.4
cm3) was added Et2NH (0.80 cm3) at 0 °C. The reaction mixture was warmed to room temperature
and stirred for 1.5 h. The reaction mixture was concentrated under reduced pressure and the residue
was purified by reverse-phase preparative HPLC (59% CH3CN in 0.1% TFA solution) to give linear
peptides 6a (29.6 mg, 30% from 33) and 6b (24.0 mg, 24% from 33) both as a colorless powder.
6a: []27D –46.8 (c 0.89, CHCl3); IR (neat) max/cm-1: 1645 (C=O); 1H NMR (500 MHz, CD3CN, 1:1
mixture of rotamers) δ: 0.77 (1.5H, d, J 7.0), 0.81-0.91 (18H, m), 0.96 (1.5H, d, J 3.1), 0.97 (1.5H, d,
J 3.1), 1.06-1.22 (4.5H, m), 1.26-1.33 (3H, m), 1.37 (3H, d, J 7.3), 1.41-1.53 (2H, m), 1.74-1.84 (5H,
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m), 1.96-2.01 (1H, m), 2.07 (1.5H, s), 2.08 (1.5H, s), 2.20-2.38 (3H, m), 2.75-2.86 (4.5H, m), 3.00-
DOI: 10.1039/C6OB01583B
3.12 (6.5H, m), 3.53 (0.5H, d, J 16.4), 3.70-3.71 (1H, m), 4.00 (0.5H, d, J 18.0), 4.05-4.09 (0.5H, m),
4.17 (0.5H, d, J 18.0), 4.24 (0.5H, d, J 16.4), 4.31-4.36 (0.5H, m), 4.48-4.50 (0.5H, m), 4.53 (0.5H,
d, J 5.0), 4.55 (0.5H, d, J 5.0), 4.62 (0.5H, d, J 5.0), 4.64 (0.5H, d, J 5.0), 4.73-4.76 (0.5H, m), 4.87-
4.89 (1H, m), 4.95 (0.5H, d, J 3.4), 4.96 (0.5H, d, J 3.4), 5.19 (0.5H, q, J 7.3), 5.24 (0.5H, q, J 7.3),
5.43-5.46 (0.5H, m), 5.63 (0.5H, dd, J1 9.9, J2 5.7), 6.82-6.89 (1H, m), 7.15-7.28 (6H, m), 7.64 (2H,
br s); 13C NMR (125 MHz, CD3CN) δ: 10.2 (2C), 11.1, 11.3, 11.9 (2C), 12.7 (2C), 13.0, 13.1, 14.2
(2C), 14.3 (2C), 14.8 (3C), 14.9, 15.5, 15.7, 16.0, 16.1, 20.8 (2C), 24.9, 25.1, 26.8 (2C), 29.3, 29.4,
30.5, 31.5, 32.5 (2C), 34.5, 34.6, 35.4, 35.6, 35.7, 36.8 (2C), 37.0 (3C), 37.2, 37.3 (2C), 37.7, 48.0,
48.6, 52.0, 52.3, 53.5 (2C), 53.7, 55.1, 55.3, 56.0, 73.6 (2C), 75.4, 75.6, 76.5 (2C), 78.9 (2C), 127.4,
127.7, 128.9, 129.0, 129.1 (2C), 129.2 (2C), 130.2 (2C), 130.3 (2C), 137.5, 137.8, 142.4 (2C), 168.3,
168.5, 168.9, 169.5, 170.0, 170.4, 171.2, 171.7, 172.2, 172.3, 172.5 (2C), 173.4, 174.0; HRMS (ESI)
calcd for C48H80N5O11S (MH+): 934.5570; found: 934.5567.
1
6b: []28 –23.5 (c 1.00, CHCl3); IR (neat) max/cm-1: 1648 (C=O); H NMR (500 MHz, CD3CN,
D
3:3:3:1 mixture of rotamers) δ: 0.74 (0.9H, d, J 6.9), 0.78-0.98 (21.9H, m), 1.05 (1.2H, d, J 6.9), 1.08-
1.51 (11H, m), 1.75-1.90 (5H, m), 1.97-2.02 (1H, m), 2.05-2.06 (2.7H, m), 2.09 (0.3H, s), 2.19-2.45
(3H, m), 2.72 (0.3H, s), 2.81-2.86 (4.8H, m), 2.89-2.91 (2H, m), 2.93 (0.3H, s), 2.95 (0.3H, s), 3.00-
3.09 (1.8H, m), 3.12-3.18 (1.5H, m), 3.43 (0.1H, d, J 16.1), 3.55-3.56 (0.1H, m), 3.61-3.76 (1.6H, m),
4.09-4.16 (0.6H, m), 4.25-4.36 (1.3H, m), 4.45-4.48 (0.4H, m), 4.52-4.64 (1.9H, m), 4.70-4.73 (0.3H,
m), 4.79-5.00 (3H, m), 5.08-5.12 (0.7H, m), 5.26 (0.3H, dd, J1 9.3, J2 6.0), 5.33 (0.3H, dd, J1 11.1, J2
4.4), 5.46 (0.3H, dd, J1 11.1, J2 4.8), 5.52 (0.1H, dd, J1 9.5, J2 6.4), 6.84-6.91 (1H, m), 6.97 (0.3H, d,
J 9.2), 7.07-7.26 (5H, m), 7.42 (0.3H, d, J 7.3), 7.69 (0.4H, d, J 6.7), 8.11 (2H, br s); 13C NMR (125
MHz, CD3CN) δ: 10.3 (3C), 10.6, 12.0 (2C), 12.2 (2C), 12.8, 12.9, 13.2 (3C), 13.7, 14.2 (2C), 14.4
(2C), 14.6 (2C), 14.7, 14.8, 14.9 (2C), 15.1, 15.2, 15.9 (2C), 16.1 (2C), 16.2, 16.3, 20.8, 20.9, 26.8
(2C), 26.9 (2C), 27.0 (2C), 27.4, 29.4, 29.5(2C), 29.6, 30.4, 30.5, 30.6, 31.1, 31.9, 32.6, 32.7, 33.6,
34.5, 34.6, 34.7, 35.2, 35.3, 35.4, 35.5, 35.6, 35.9, 36.5, 36.8, 37.0 (4C), 37.1, 37.2, 37.4 (2C), 38.1,
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38.4, 48.0 (2C), 48.6 (2C), 52.5, 52.6, 53.0, 53.2, 54.0, 54.3 (2C), 54.7, 54.8, 55.0, 5D6O.4I: ,105.1703.19/,C65O8B.011,583B
73.5, 75.3 (2C), 75.6 (2C), 75.9, 76.1 (2C), 78.6, 79.0, 79.6, 79.7, 127.5 (2C), 127.6, 129.1 (3C),
129.2 (3C), 129.3, 130.2 (2C), 130.3 (3C), 130.4 (2C), 137.5, 137.8, 138.1, 138.3, 141.4, 141.5, 141.9,
142.2, 167.9, 168.1, 168.2, 168.3, 168.7, 169.6, 170.1, 170.2, 170.3 (2C), 170.8, 171.2 (2C), 171.3,
171.4, 171.8, 172.0, 172.2 (2C), 172.5, 173.4, 174.4, 174.7; HRMS (ESI) calcd for C48H80N5O11S
(MH+): 934.5570; found: 934.5580.
Odoamide (5a). To a stirred solution of 6a (17.8 mg, 0.017 mmol), HOAt (11.6 mg, 0.085 mmol)
and collidine (0.067 cm3, 0.51 mmol) in DMF (17.0 cm3) was added HATU (64.6 mg, 0.17 mmol) at
room temperature. After stirring for 5 h, the reaction mixture was concentrated under reduced
pressure, and EtOAc and 1N HCl were added to the residue. The whole was extracted with EtOAc
and the extract was washed with brine, aqueous saturated NaHCO3 and brine, dried over MgSO4. The
filtrate was concentrated under reduced pressure and the residue was purified by flash
chromatography over silica gel with hexane–EtOAc (1:1 to 0:1) to give the corresponding cyclic
peptide. To a stirred solution of the above cyclic peptide in THF/H2O (4:1, 0.566 cm3) were added
2,6-lutidine (0.0394 cm3, 0.34 mmol) and AgNO3 (115.5 mg, 0.68 mmol) at room temperature. The
reaction mixture was warmed to 70 °C and stirred for 4 h. The mixture was filtered through Celite,
and 1N HCl was added to the filtrate. The whole was extracted with EtOAc and the extract was
washed with H2O, aqueous saturated NaHCO3 and brine, dried over MgSO4. The filtrate was
concentrated under reduced pressure and the residue was purified by reverse-phase preparative HPLC
(72% CH3CN in H2O) to give odoamide (5a) (12.4 mg, 85%) as a colorless powder: []28D –15.8 (c
1.14, CH3OH); IR (neat) max/cm-1: 3305 (OH), 1645 (C=O); 1H NMR (500 MHz, CD3OD) δ: 0.83-
0.96 (21H, m), 1.04-1.12 (4H, m), 1.19-1.26 (1H, m), 1.29-1.40 (4H, m), 1.42 (3H, d, J 6.9), 1.45-
1.55 (1H, m), 1.57-1.67 (1H, m), 1.78-1.87 (3H, m), 1.90 (3H, s), 2.01-2.04 (1H, m), 2.12-2.16 (1H,
m), 2.20-2.28 (1H, m), 2.85-2.95 (4H, m), 3.01-3.06 (4H, m), 3.30 (3H, s), 3.56 (1H, d, J 18.3), 3.74-
3.76 (1H, m), 3.94 (1H, q, J 6.9), 4.19 (1H, d, J 18.3), 4.49 (1H, q, J 6.9), 4.86-4.89 (2H, m), 5.05
20

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(1H, d, J 6.3), 5.45 (1H, dd, J1 10.3, J2 5.2), 7.12-7.20 (5H, m), 7.31-7.32 (1H, m);DOCI: 10N.10M39R/C6(O1B2015583B
MHz, CD3OD) δ: 10.0, 11.7, 12.0, 12.1, 13.1, 13.8, 14.5, 14.6, 15.6, 16.0, 21.6, 24.7, 27.4, 30.5 (2C),
35.8, 35.9, 36.6, 37.6, 37.8, 38.5, 39.4, 41.3, 46.4, 52.6, 54.7, 55.0, 60.3, 71.5, 77.6, 79.2, 127.4,
128.6, 129.1 (2C), 130.6 (2C), 138.4, 146.8, 170.4, 171.3, 172.5, 172.7, 172.8, 173.0, 174.9; HRMS
(ESI) calcd for C46H73N5NaO10 (MNa+): 878.5250; found: 878.5254.
Growth Inhibition Assay
A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented
with 10% (v/v) fetal bovine serum at 37 °C in a 5% CO2-incubator. Growth inhibition assays using
A549 cells were performed in 96-well plates (BD Falcon). A549 cells were seeded at 500 cells well-
1
in 0.050 cm3 of culture media, respectively, and were cultured for 6 h. Chemical compounds in
DMSO were diluted 250-fold with the culture medium in advance. Following the addition of 0.040
cm3 of the fresh culture medium to the cell cultures, 0.030 cm3 of the chemical diluents were also
added. The final volume of DMSO in the medium was equal to 0.1% (v/v). The cells under chemical
treatment were incubated for further 72 h. The wells in the plates were washed twice with the cultured
medium without phenol-red. After 1 h incubation with 0.100 cm3 of the medium, the cell culture in
each well was supplemented with 0.020 cm3 of the MTS reagent (Promega), followed by incubation
for additional 40 min. Absorbance at 490 nm of each well was measured using a Wallac 1420 ARVO
SX multilabel counter (Perkin Elmer). Three experiments were performed per condition and the
averages of inhibition rates in each condition were evaluated to determine IC50 values using the
GraphPad Prism software.
Acknowledgements
This work was supported by Grants-in-Aid for Scientific Research from JSPS, Japan (24659004
and 15J05499); Platform for Drug Discovery, Informatics, and Structural Life Science from MEXT,
Japan; and Takeda Science Foundation. M.K. is grateful for JSPS Research Fellowships for Young
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Scientists.
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21. Two hydroxy group configurations in 12c and 12d were determined by the NMR analysis of the
DOI: 10.1039/C6OB01583B
corresponding acetonides.
22. (5R)-Hydroxy ester 26a is the substrate for the synthesis of (5S)-hydroxy ester 12a (see
supplementary information).12
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27. Among several conditions investigated for coupling of peptide 9, EDCI-HOAt provided the
desired 6a in higher chemical yield; however, significant epimerization at the C-terminal L-Ile
occurred.
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Figure 1. Structures of aurilide-class depsipeptides.
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Figure 2. Structures of odoamide (5) (A) and the polyketide substructures in 5 (B).
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natural 5
synthetic 5a
Figure 3. Comparison of the 1H NMR spectra between the natural compound 5 and the synthetic 5a
(in CD3OD).
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natural 5
synthetic 5a
Figure 4. Comparison of the 13C NMR spectra between the natural compound 5 and the synthetic 5a
(in CD3OD).
29